Because the conversion efficiency of fluorophores is extremely low, fluorescence microscopy is an extremely inefficient process in which light source-to-detector efficiency may be in the range of parts or a fraction of a part per billion. Another limitation of fluorescence imaging is that the intensity of an illumination source needs to be limited to avoid destruction of the sample or so-called “photo bleaching” in which the capability of the fluorophores to fluoresce is diminished; even before the condition of photo bleaching is reached, the behavior of most fluorophores becomes significantly non-linear or unpredictable, imposing further optical constraints. Numerous non-optical constraints also affect the practicality of the fluorescent microscope design, such as the acceptable duration of a scan of an array, the reliability of the data, the cost of the biochip, the processing complexity and the cost of the scanner.
The state of the art of fluorescence biochip imaging has accordingly been guided by the necessity for a microscope reader to have a very efficient fluorescence-emitted light capture capability. As well, a very shallow depth of field has been important so that only the very thin layer of biological material is imaged, to avoid optical noise perturbations that may be emitted from the support member under the array. This approach has lead to complex and expensive systems: epi-fluorescent scanning confocal microscope readers and cooled CCD camera-based readers that have a small field of view and require moving or tilting with respect to two axes to scan the array.
Some epi-fluorescent confocal or near confocal scanning microscopes employ high precision radiation-directing systems driven by galvanometers or motors and single point detectors such as PMTs or diodes. The text edited by Mark Schena and published by Bio Techniques Books, Natick, Mass. pp. 53–64 carries a summary description of a number of such commercial instruments. Others are also described in U.S. Pat. No. 3,013,467 (Minsky); U.S. Pat. No. 5,459,325 (Hueton); U.S. Pat. No. 5,981,956 (Stern); U.S. Pat. No. 5,895,915 (DeWeerd); U.S. Pat. No. 5,585,639 (Dorsel); U.S. Pat. No. 5,646,411 (Kain); U.S. Pat. No. 5,672,880 (Kain); U.S. Pat. Nos. 6,335,824; 6,201,639 and 6,185,030 (Overbeck).
Examples of fluorescence microscopes that use a CCD array imager as a detector are shown in the Handbook of Biological Confocal Microscopy edited by James Pawley, Plenum Press, 1989 and 1995. Others can be found in U.S. Pat. No. 5,900,949 (Sampas).
In total, the low efficiency of the fluorescent conversion and the other factors mentioned have lead to slow and costly reading of conventional biochips whether by high accuracy scanning of the confocal microscope or by the high cost system of a cooled CCD-based camera associated with a high accuracy scanning mechanism. Such expensive systems have mainly been employed in academic studies and in large efforts directed to drug discovery. No practical way has emerged to enable the technology to be adapted to much lower cost uses such as in medical clinics and diagnostic laboratories, in veterinary medicine, in dealing with agricultural crop diseases and food and water processing, and in lower level educational and investigational laboratories.
An object of the invention is to provide an improved fluorescence imaging approach, and in particular a diagnostic tool that is low-cost and highly effective, useful in direct patient diagnosis and treatment in medicine, as well as for other purposes such as those mentioned.
The invention employs surface light effects to concentrate the illumination in the vicinity of the plane of the sample array. In this way the excitation energy density can be enhanced at the surface of the sample relative to objects at other depths, and imaging can be done without requiring that the imaging system, itself, have a very shallow depth of field. Numerous patents exemplify application of this general technology to experimental microscopy. Examples are U.S. Pat. Nos. 5,633,724; 5,351,127 and 5,437,840 (King) and U.S. Pat. No. 5,341,215 (Seher) and European Patent Application 93304605.4 (EP 0575 132 A1) (King) as well as trade journal articles such as Photonics Spectra, February 2000, pages 24–26. The technique has been described in the Conference on Advances in Fluorescence Sensing Technology IV, 1999, Vol. 3602 pp. 140–148 and pp. 94–101; the Proceedings of SPIE Vol. 4252 pp. 36–46, SPIE Vol. 2928 pp. 90–109 and SPIE Vol. 3858 pp. 59–71, and the book Internal Reflection Spectroscopy by F. M. Mirabella Jr. and N. J. Herrick. See also U.S. Pat. Nos. 5,910,940, 5,754,514 and 5,666,197 (Guerra) and U.S. Pat. No. 6,078,705 (Neuschafer et al.), from different fields.
The potential of surface wave techniques for illuminating and detecting specific binding analytes is also well documented. A number of the techniques proposed use prisms or gratings to induce evanescent fields. Other related techniques such as Surface Plasmon Resonance (SPR) couple evanescent incident radiation into a mode generated between a thin metal layer, such as gold or silver, and a dielectric layer such as silicon or phosphate glasses or silane. Such techniques have been described in U.S. Pat. Nos. 5,830,766 and 5,631,170 and PCT WO90/06503 (Attridge).
A preferred technique to create an evanescence fluorescence-enhancing surface wave described in certain of the above references is to illuminate the substrate at a defined illumination region, via an intermediate support. The light arrives at the surface at a suitable angle to induce an evanescent wave on the surface. To excite the sample, the energy then travels laterally along the surface to a separately defined sample region where the sample is excited. Often a large 90 degree prism member has been employed which is coupled to a separate member carrying the biology. Varying the angle of the incident light permits the accommodation of a range of illumination wavelengths. A conventional microscope has then been used to inspect the fluorescence. By this technique, illumination of the sample has been enhanced without the penalty of incident light being reflected into the objective of the reader. Often a fluid coupling agent between the mated optical parts is required. A variation on this technique has used a grating at an illumination region separate from the imaging region, and the angle of illumination of the grating has been tuned to maximize the signal, see Review on Fluorescence-Based Planar Wave Guide Biosensors, Duveneck et al., Vol. 3858, 1999. In another field a large transparent optical block has been employed to couple light to a sample at various angles for sectioning the sample at various levels, see e.g. U.S. Pat. No. 6,255,642 (Cragg et al.)
Applications exist where such previously designed surface wave systems may be justified, but these designs have not proven suitable for low-cost clinical usage and the like.
One previously proposed substrate for imaging a fluorescing array has been a microscope slide having an interference grating buried under a thin layer of high index glass. In that example the grating was arranged to reflect normal incident light that has not been absorbed by the sample (a very large fraction) at a suitable angle to induce an evanescent wave at the sample. The intensity of the evanescent wave can be more than an order of magnitude greater than that of the original incident light beam, but, because gratings reflect normal incidence beams at different angles for different wavelengths, to operate beneficially, each slide was generally restricted to its design wavelength.
U.S. patents disclosing other use of gratings include U.S. Pat. No. 5,822,472 (Danieizik et al.) and U.S. Pat. Nos. 6,078,705 and 6,289,144 (Neuschafer et al.) In these and in other cases the array to be imaged has been incorporated in a flow cell arrangement, see also for example U.S. Pat. Nos. 5,166,515; 5,344,784; 5,631,170 and 5,830,766 (Attridge); and U.S. Pat. No. 4,857,273 (Stewart).
Prior art CCD-based, fluorescent, conventional or confocal scanning microscope systems can provide high quality images of material located on the top surface of a support. But in their compromise between depth of field, energy collection efficiency, laser power, damaging of the sample by photo bleaching, capture time requirements and cost and the precision and complexity associated with establishing evanescence light concentration, high cost of the support, uncertainties caused by operating in a non-linear region of the fluorophores, etc., they have not been altogether satisfactory.
It is well known that evanescent illumination of a biochip has had the potential to offer a much higher signal than conventional illumination, such that a CCD-based imaging system can be used to acquire the image information on the biochip without loss of data. The apparent requirements and cost of prior proposals to reliably induce evanescence, however, has apparently impeded commercialization of the techniques.
The present invention provides low-cost, robust and wave-length versatile systems and techniques incorporating surface wave technology that are foreseen to enable a breakthrough in the technology.